The preservation and storage of biologically active materials, viruses, cells and small multicellular specimens is important for many applications, including research, food, microbiological, pharmaceutical and healthcare industries as well as for agriculture.
One of the most important criteria in evaluating the efficacy of practically all preservation techniques is how stable the resultant product is. It is well known that, in an aqueous phase, viral and bacterial vaccines, therapeutic proteins and other biologicals instantly lose activity during storage at ambient temperatures (AT). For example, according to Dr. Truong (as described in a U.S. patent application No. 20030219475), enveloped viruses—such as live influenza virus manufactured from egg allantoid fluid—loose one log of potency, defined as Tissue Culture Injectious Dose (TCID50), in less than two to three weeks when stored under refrigerated temperature, i.e. approximately 4 degrees Celsius. At room temperature conditions (approximately 25 degrees Celsius) and at warmer temperatures such as 37 degrees Celsius, the virus looses such potency in a matter of days to hours, respectively. An ability to store dehydrated biologically active materials at ambient temperatures for extended periods of time carries with it enormous benefits. Dehydrated reagents, materials, and biologicals are characterized by significantly reduced weight. In addition, they require less space for storage and, at the same time, offer increased stability.
Currently, measles vaccines are preserved by freeze-drying. Because the freeze-dried vaccines are stable only at near 0° C. temperatures, the measles vaccines need to be refrigerated at all times. World Health Organization (WHO) has estimated that just the maintenance of the existing “cold chain” in economically challenged countries (ECC) costs over $200 million annually. In addition, many rural areas do not have refrigeration at all, which makes it either practically impossible or very costly to administer existing measles vaccines in such areas.
Availability of stable at ambient temperature and more potent vaccines against MMR, tuberculosis, flu and other diseases will have an enormous impact on human health worldwide. Storage at ambient temperatures would eliminate the need for a cold chain, a costly and challenging logistical problem in many parts of the world, especially those parts where many of these vaccines are needed the most.
Existing methods for manufacture and storage of live vaccines require improvement for two major reasons. First, during manufacture, the vaccine is typically lyophilized or freeze-dried. Conventional freeze-drying is very damaging to cellular components and other biologicals, which, typically, results in reduced viability of the vaccine by a log or more. Second, conventionally freeze-dried products are stable only at or near 0° C., which requires that the vaccine be refrigerated from the time it is manufactured until the time it is administered. Hence, a so-called “cold chain” needs to be maintained during storage and transportation. In many instances, including transportation within the developing world and remote areas, refrigeration is either unavailable or problematic. Even if refrigeration is available, it significantly increases the costs of storage and transportation. Thus, development of a method for stabilizing vaccines so that they can be stored and transported at ambient temperatures is an important objective of this invention. So far, notwithstanding the attempts of numerous researchers, no such methods have been developed using freeze-drying, and the most common methods based on freeze-drying have failed to eliminate the need for the “cold chain.”
Stabilization by Vitrification (Glass Formation)
While for a limited amount of time (several days), stabilization of sensitive biologicals, including biological macromolecules, viruses and cellular items, can be achieved in a liquid state, the long-term (several months, several years or more) stabilization of the biologicals requires arresting molecular mobility to stop degradation processes during storage. This can be achieved by vitrification, which is a transformation from a liquid into a highly immobile, noncrystalline, amorphous solid state, known as the “glass state.”
A “glass state” is an amorphous solid state, which may be achieved by supercooling of a material that was initially in a liquid state. Diffusion in vitrified materials (i.e., glasses) occurs at extremely low rates (e.g., microns/year). Consequently, chemical and biological changes requiring the interaction of more than one moiety are practically completely inhibited. Glasses normally appear as homogeneous, transparent, brittle solids, which can be ground or milled into a powder. Above a temperature known as the glass transition temperature Tg, the viscosity drops rapidly and the material transforms from a glass state into what is known as a deformable “rubber state.” As the temperature increases, the material transitions into a liquid state. The optimal benefits of vitrification for long-term storage may be secured only under conditions where Tg is greater than the storage temperature.
Although scientists still dispute thermodynamic models that explain the transformation of highly supercooled liquids, or supersaturated solutions, into the “glass state” during cooling, vitrification has been broadly used to preserve biological and highly reactive chemicals. The basic premise of vitrification is that all diffusion limited physical processes and chemical reactions, including the processes responsible for the degradation of biological materials, stop in the glass state. This premise is based on Einstein's theory that establishes the relation between viscosity and diffusion. In general terms, glasses are thermodynamically unstable, amorphous materials that are mechanically stable at their very high viscosity (1012-1014 Pa·s.). A typical liquid has a flow rate of 10 m/s compared to 10−14 M/s in the glass state.
For many years, it has been well-known that biologicals can be preserved at −196° C. Tg for pure water is about −145° C. If ice crystals form during cooling, the solution that remains unfrozen in the channels between ice crystals will vitrify at Tg′, which is higher than Tg for pure water Biologicals that are rejected in the channels during ice growth will be stable at temperatures below Tg′.
The damaging effect of cryopreservation is mostly associated with freeze-induced dehydration, change in pH, increase in extracellular concentration of electrolytes, phase transformation in biological membranes and macromolecules at low temperatures, and other processes associated with ice crystallization. Potential cryodamage is a drawback in the methods that rely on freezing of biologicals. This damage could be decreased by using cryoprotective excipients (protectants), e.g., glycerol, ethylene glycol, dimethyl sulfoxide (DMSO), sucrose and other sugars, amino acids, synthetic, and/or biological polymers, etc.
Biologicals can be stabilized at temperatures substantially higher than −145° C. if they are placed in concentrated preservation solutions with high Tg. For example, for a solution that contains 80% sucrose, Tg is about −40° C. A solution that contains 99% sucrose is characterized by Tg of about 52° C. The presence of water in a sample results in a strong plasticizing effect, which decreases Tg. The Tg is directly dependent on the amount of water present, and may, therefore, be modified by controlling the level of hydration—the less water, the higher the Tg. Therefore, the specimens (to be vitrified at an ambient temperature) must be strongly dehydrated by drying. However, drying can be damaging to biologicals. Therefore, to stabilize biologicals at a room temperature and still preserve their viability and functions, they need to be dried in the presence of a protective excipient (i.e., protectant) or a combination of excipients, which have a glass transition temperature Tg higher than the room temperature.
There are at least two aspects of stabilization in a dry state that should be optimized to arrive at a method for preserving biologicals that results in a preserved material suitable for a long-term storage at ambient temperatures: (1) it is important to formulate an effective preservation solution that will not crystallize during the drying process and, at the same time, will reliably protect the biological from damage that may be caused by dehydration stress; and (2) the dehydration method must allow for an efficient and scalable way to dry the subject material.
Prior art teaches several methods for providing enhanced-stability preparations of labile biological materials in dehydrated form: freeze-drying, vacuum or air-drying by evaporation (preservation by evaporation), and preservation by foam formation.
Preservation by Evaporation
The application of drying for preservation of biopharmaceuticals was recorded several centuries ago. It was reported then that the sloe berries juice “may be reduced by gentle boiling to a solid consistence, in which state it will keep the year round.” At the beginning of the last century, many scientists performed comparisons between the stabilizing effects of evaporation from the liquid state vs. freeze-drying. As a result, it has been established that activity of biologicals dried by evaporative drying of small drops is comparable to and in many cases even better than activity of freeze-dried samples. For example, it has been shown that labile enzymes (luciferase and isocitric dehydrogenase) can be preserved by evaporative drying for more than a year at 50° C. without any detectable loss of activity during drying and subsequent storage at 50° C. (Bronshtein, V., Frank, J. L., and Leopold, A. C. (1996). Protection of Desiccated Enzymes by Sugars. In: “Cryo 96 program”, Abstract 22 of a Paper Presented at the 33rd Annual Meeting of the Society for Cryobiology, Indianapolis, Ind.; Bronshtein, V., and Leopold, A. C. (1996) Accelerated aging of dried luciferase and isocitrate dehydrogenase. Effect of sugar/enzyme mass ratio. In: “Cryo 96 program”, Abstract 23 of a Paper Presented at the 33rd Annual Meeting of the Society for Cryobiology, Indianapolis, Ind.) Unfortunately, because dehydrated solutions containing protectors become very viscous, it takes long periods of time to evaporate water even from small drops of a solution. Therefore, until now, industrial applications have utilized freeze-drying methods because evaporative drying is a diffusion-limited process that is not scalable to industrial quantities.
Freeze-Drying (FD)
Freeze-drying, or lyophilization, has been known and applied to preserve various types of proteins, viruses, and cells, including RBCs, platelets, and microorganisms. FD consists of two major steps: primary drying and secondary drying.
Freeze-drying can be used to produce stable biologicals in industrial quantities. However, as a practical matter, it is very difficult (if not impossible) to develop a continuous load freeze-drying process for cost-effectively making industrial quantities of stable biologicals. In addition, it is very difficult to execute freeze-drying as a barrier process (i.e., a process where the operator is sufficiently separated from the material being preserved) in both vial for unit dose production and in bags, trays or other containers for bulk production. New methods are necessary to satisfy all requirements of industrial production.
Primary Freeze-Drying
The limitations of freeze-drying, as described above, result in part from a need to utilize low pressure (or high vacuum) during a freeze-drying process. A high vacuum is required because the temperature of the material during the primary freeze-drying should be below its collapse temperature, which is approximately equal to Tg′. At such low temperatures, the primary drying takes many hours (sometimes days) because the equilibrium pressure above ice at temperatures below −25° C. is less than 0.476 Torrs. Therefore, a new process must allow for shorter production times.
The low vacuum pressure used in the existing freeze-drying methods limits the amount of water that can be removed from a drying chamber to a condenser per one unit of time. Therefore, it is impossible to build an industrial manifold freeze-dryer with a volume of material to be dried in each chamber equal to several liters or more, which is necessary for an industrial scale production. New methods are necessary to allow for efficient industrial scale production of sufficiently large amounts of preserved biologicals.
In addition, such low water vapor pressures limit the selection of films that can be used to isolate the target material in bags from the environment of the chamber during the freeze-drying process. Currently the industry uses Lyoguard trays covered with Gore membranes. Gore membranes are made with pores to be permeable to water vapor, which is necessary for any drying process. Because of the presence of pores, Gore membranes are also permeable by some viruses.
Primary freeze-drying is performed by sublimation of ice from a frozen specimen at temperatures close to or below Tg′ that is a temperature at which a solution that remains not frozen between ice crystals becomes solid (vitrifies) during cooling. According to conventional beliefs, performing freeze-drying at such low temperatures is important for at least two reasons.
The first reason for which freeze-drying at low temperatures (i.e., below Tg′) is important is to ensure that the cake remaining after ice removal by sublimation (primary drying) is “solid” and mechanically stable, i.e., that it does not collapse. That is a valid reason. Keeping the cake in a mechanically stable “solid” state after primary freeze-drying is important to ensure effective reconstitution of the freeze-dried material. Several methods were proposed to measure the Tg′ for a specific material. These methods rely on different interpretations of the features that can be seen in DSC (Differential Scanning calorimeter) thermograms. The most reliable way to determine Tg′ is based on an evaluation of the temperature at which ice begins to melt and the concentration of water remaining unfrozen (Wg′) during slow cooling. The following relevant data have been reported:Sucrose: −38.8° C.<Tg′<−37.55° C., and 18.76 wt %<Wg′=1−Cg′)<19.42 wt %;Glucose: −59.9° C.<Tg′<−49.37° C. and 18.76 wt %<Wg′=1−Cg′)<19.42 wt %;Sorbitol: −54.44° C.<Tg′<−52.03° C., and 18.76 wt %<Wg′=1−Cg′)<19.42 wt %.
For a solution of bovine serum albumin in water Tg′ is −20° C. and Wg′ is 20 wt %. For this reason, the primary freeze-drying should be performed at temperatures below −20° C. in a temperature range called Intermediately Low Temperatures (ILT), which is approximately between −25° C. and −50° C.
The second reason typically advanced to support the importance of freeze-drying at low temperatures (i.e., below Tg′) is that the survival rate of biologicals after freeze-drying is higher if the primary freeze-drying is performed at lower temperatures. Two principal arguments are typically used to support this notion. The first one is that drying at lower temperatures is beneficial because it “ . . . slows the kinetics of degradation reactions” (see for example, U.S. patent application Ser. No. 10/412,630). The second argument used to support the connection between freeze-drying at low temperatures and the survival rate of biologicals is that freeze-drying induced damage occurs primarily during the secondary drying after ice lyophilization is completed. (Webb, S. D. Effect of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-gamma. J Pharm Sci, 2003, 92 (4):715-729).
However, both of the above arguments are erroneous because the decrease in reaction rates expected from the Arrhenius kinetics is applicable only to unfrozen solutions. The reaction rates actually increase in frozen solutions because ice crystals concentrate solutes and biologicals in the channels remaining unfrozen between the crystals. In theory and in practice, freeze-drying (“FD”) is very damaging for sensitive biologicals. Strong FD-induced injury occurs during both freezing (formation of ice crystals) and the subsequent equilibration of the frozen specimens at intermediately low temperatures during ice sublimation. Well-known factors that cause cell damage during freezing include: freeze-induced dehydration, mechanical damage of cells during ice crystallization and recrystallization, phase transformation in cell membranes, increasing electrolyte concentration and others. However, possibly the principal factor that damages frozen biologicals is the occurrence of a large pH change in the liquid phase that remains unfrozen between ice crystals. This abnormal pH change, which can be as large as 5 units (i.e., pH>12), is associated with crystallization hydrolysis, as described in “Freezing Potentials Arising on Solidification of Dilute Aqueous Solution of Electrolytes.” V. L. Bronshteyn, A. A. Chemov, J. Crystal Growth 112: 129-145 (1991).
Crystallization hydrolysis occurs because ice crystals capture positive and negative ions differently. This creates a significant (about 107 V/m) electrical field inside ice crystals. Neutralization of this electrical field occurs due to electrolysis inside the ice crystals at a rate proportional to the constant of water molecule dissociation in ice. This neutralization results in a change of the pH of the liquid that remains between the ice crystals. The damaging effect of crystallization hydrolysis can be decreased by reducing the surface of ice that forms during freezing and by increasing the volume of the liquid phase that remains between the ice crystals. This remaining liquid also reduces the damaging effect of (i) the increasing electrolyte (or any other highly reactive molecules) concentration and (ii) the mechanical damage to cells between the ice crystals. The increase of the liquid between the ice crystals can be achieved by (i) increasing the initial concentration of of protectants added before freezing, and (ii) by decreasing the amount of ice formed in the sample.
Avoiding freezing to temperatures equal to Tg′ or below (at which freeze-drying is typically performed) will allow to significantly reduce the amount of damage in the preserved biological. Therefore, a new method that allows a preservation of biologicals without subjecting the biologicals to temperatures near or below Tg′ will significantly improve the quality of the preserved material.
Secondary Freeze-Drying
After the removal of ice by sublimation (primary drying) is complete, the sample may be described as a porous cake. Concentration of water in the sample at the end of primary drying is above the concentration of water Wg′ that remains unfrozen in the glassy channels between ice crystals at a temperature below Tg′. The data presented above show that Tg′ strongly depends on the composition of the solution, while for the majority of solutes Wg′ is about 20 wt %. At such high water concentrations, the glass transition temperature of the cake material is below the primary freeze-drying temperature, and/or significantly below −20° C. Secondary drying is performed to remove the remaining (about 20 wt %) water and increase the glass transition temperature in the cake material. As a practical matter, secondary drying cannot be performed at Tg′ or lower temperatures because diffusion of water from a material in a glass state is extremely slow. For this reason, secondary drying is performed by heating the cake to a drying temperature Td that is higher than the glass transition temperature Tg of the cake material at a given moment. If during the secondary drying step, Td is substantially higher than Tg, the cake will “collapse” and form a very viscous syrup, thereby making standard reconstitution impossible. Therefore, the collapse of the cake is highly undesirable.
The collapse phenomenon, which is kinetic by nature, has been extensively discussed in the literature. The rate of the collapse increases as the viscosity of the cake material decreases. To avoid or bring the collapse process to a negligible scale, Td is kept close to Tg during the secondary drying, thereby ensuring that the viscosity of the cake material is high and the rate of the collapse slow.
During secondary drying, removal of water occurs through evaporation from the internal and external surfaces of the cake and is limited mostly by the rate of water diffusion inside the cake material. For this reason, secondary drying also takes many hours. Water diffusion inside the very viscous cake material during the secondary drying step is a very slow process that creates high gradients of water concentration inside the cake material. Therefore, at the end of the secondary drying step, Tg of the cake material is normally still far below the maximum Td used during the secondary drying. In many cases, this explains why biologicals are not stable after preservation by freeze-drying.
To simplify the analysis, the characteristic time t of this process can be estimated using equation t=h2/D, where h is a thickness of the specimen and D is the water diffusion coefficient. For water, D approximately equals 10−5 cm2/sec. Given D=10−5 cm2/sec, it will take only about 10−3 sec to dry a small specimen with a thickness of 1 μm. However, D quickly decreases as the extent of dehydration, vitrification temperature (Tg), and, viscosity in the specimen increase. If Tg can be increased during dehydration up to the temperature at which drying is performed, D will decrease (while viscosity will increase) approximately fourteen (14) orders of magnitude or more. As a result, the time required to remove water from a 1 μm specimen will be close to ten thousand (10,000) years. Therefore, as a practical matter, the glass state can only be achieved by cooling (at a constant pressure) and not by drying. For the same reason, when drying a biological solution, one cannot achieve a vitrification temperature Tg higher than the temperature Td at which the drying is performed. This is a basic phenomenon that has been overlooked by many scientists who do not appreciate how slow the drying is at temperatures close to Tg or below. For example, Roser et al. (U.S. Pat. No. 5,762,961), Schebor et al. (Journal of Food Engineering, 30, 269-282, 1996), Sun et al. (Physiologia Plantarum 90, 621-628, 1994), and many other researchers have reported values of Tg much higher than the temperature at which the material was dried Td. In these publications, to determine Tg, the authors must have misinterpreted their test results obtained by DSC (Differential Scanning calorimeter) devices. A more reliable measurement of Tg should be performed by measuring the onset of thermally stimulated polarization (or depolarization) or the onset in specific heat change during a transition from the glass to liquid state.
Preservation by Foam Formation (PFF)
For more than fifty years, freeze-drying has been a dominant method for preservation of labile biologicals. This choice has been based on a conventional belief that freeze-drying is the only scalable (industrial) technology that can allow for a preservation of labile biologicals in a dry state. Other known methods, such as spray drying, drying with supercritical fluids, and other scalable methods of desiccation fail to preserve sensitive biologicals. During spray drying, small drops of biological or pharmaceutical suspensions or solutions are sprayed into a hot (above 100° C.) inert gas or air atmosphere, where they are quickly dried into a powder. The high temperatures used in this method cause unacceptable damage to sensitive biologicals. In addition, this method may not provide for sufficiently dehydrated biologicals and, depending upon a specific residual moisture requirements for product stability, additional drying by other means, such as vacuum shelf drying, may be required.
Approximately half a century ago, it was demonstrated by Annear that concentrated solutions and biological liquids that contained sugars or amino acids could be dried by foaming syrup under a vacuum. Annear applied this process to preserve several bacteria in a dry state. To obtain a syrup, Annear used sublimation and evaporation of water from the specimens. He did not believe that his process could be used for industrial applications. Later, in 1996, Roser and Gribbon (WO 96/040077) disclosed using exactly the same process to incorporate biologicals into a dry foam matrix. According to Roser and Gribbon, biological solutions should be evaporated first to obtain a “syrup” and, second, should be foamed by boiling the syrup under a vacuum. They actually defined the term “syrup” to mean a viscous solution that would foam during boiling. Thus, to foam specimens under vacuum, Annear had to obtain the syrup first by evaporation.
In 1996, a method was proposed for using the foaming process discovered by Annear to build a practical technology for preservation of sensitive biologicals in a dry state and a scalable preservation by foam formation protocol was developed (U.S. Pat. Nos. 5,766,520 and 6,306,345) These techniques, have been used to develop methods for stabilization at ambient temperatures for many bacteria, viruses, enzymes, therapeutic proteins and other molecular items. It also has been demonstrated that Annear's process can be scaled up to over 0.5-liter volumes by avoiding evaporation to obtain the syrup before the boiling begins. Since 1996, this innovative technology has been successfully applied to preserving sensitive biologicals.
After 1996, additional extensive studies have demonstrated the benefits of the PFF technology. (The PFF technology is also known as the VitriLife™ technology). Some of the results obtained after 1996 demonstrate that:
Molecular items like Amphotericin, Urokinas, Luciferase, β-Galactosidase, Ice Nucleating Protein, Taq DNA polymerase, and others can be stabilized at 37° C. or higher temperatures without any loss of activity.
Live viral vaccines from different taxonomic groups including, Herpesviridae (Bovine Rhinotracheitis), Paramyxoviridae (Measles, Bovine Respiratory Syndrome Virus (BRSV), Bovine Parainfluenza, Canine Parainfluenza, Canine Distemper), Flaviviridae (Bovine Viral Diarrhea), Parvoviridae (Canine Parvovirus), and retroviruses (MLV) can be stabilized at temperatures up to 37° C. without significant loss of activity.
Live bacterial vaccines like Salmonella choleraesuis, Salmonella typhi, Bordetella bronchiseptica, Pasteurella multocida and Pasteurella haemolytica, and many other bacteria including E. coli and L. Acidophilus can be effectively stabilized at 37° C. or higher temperatures.
At the same time, known attempts to preserve sensitive biologicals by conventional freeze-drying technology, in many cases, resulted in 10% or less survival yield and limited stability at ambient temperatures, i.e., without refrigeration. For example, survival yield of BRSV after conventional freeze-drying was less than 10% of a control sample. However, no detectable loss in the BRSV survival rate was observed in the specimens preserved by using preservation by foam formation. In 2002, the VitriLife™ technology was acquired to Avant Immunotherapeutics, Inc. (Avant).
The advantages of vitrification technology have not been fully utilized for achieving long-term stability of labile biological materials at ambient temperatures. Existing methods of ambient temperature preservation by drying are designed for laboratory scale processing of relatively small quantities of materials in unit dose vials, which makes these methods incompatible with large scale commercial operations. Technical problems related to monitoring of the glass transition temperature also have also presented obstacles to commercial implementation. While drying and vitrification technology are potentially attractive as scalable methods for long-term efficient storage of biological materials a number of problems need to be addressee before the advantages of storage in the glass state can be commercially exploited.
Despite the many benefits of the PFF (VitriLife™) technology, the technology also has some drawbacks. If one uses the approach described by Roser and Gribbon (WO 96/040077) and applies the evaporation to obtain a syrup, one will quickly find that in many cases or in a portion of vials, the boiling and foaming will not take place at all, even after an application of a high vacuum because the vapor phase cannot nucleate in a highly viscous syrup. This phenomenon makes practically impossible to validate an industrial scale PFF process developed in a lab for a specific biological. The process disclosed by Bronshtein in 1996 (U.S. Pat. No. 5,766,520) provides for a boiling step before the high viscosity of the material is achieved. The major drawbacks of that process are that it is characterized by uncontrollable eruptions of the material during boiling. These eruptions result in a portion of material splattered on the walls of the vials, which can pollute stoppers. In addition, some of such material may be released from the vials into the drying chamber. To soften the eruption during boiling and to make the boiling more gentle, it has been proposed to use two dimensional temperature/pressure application protocols that reduce overheating to an acceptable level. However, this protocol is difficult to implement and is difficult to reliably reproduce with different formulations. In many cases special processing requires to initiate nucleation of vapor bubbles (boiling) (U.S. Pat. No. 6,884,866) of the syrup obtained by evaporation.
Therefore, the PFF process is characterized by a number of significant drawbacks that severely limit its application on an industrial scale. Consequently, a new process free of the drawbacks associated with the PFF methods is necessary to improve preservation of biologicals on an industrial scale.